The formation and maintenance of single-stranded DNA (ssDNA) is an important part of many processes involving DNA. For instance, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases and this exposure of the bases on the DNA allows further processing, such as repair or replication. Assays of helicase activity are an important part of probing the related biological processes.
Although there are methods to measure ssDNA, as opposed to dsDNA, in real time assays, sensitivity and time resolution have been limited. Generally, fluorescence-based assays have provided the combination of high sensitivity and time resolution required for these types of measurement. There are several dyes that provide fluorescence signals to monitor dsDNA to ssDNA transformation [1,2], but many bind specifically in the grooves of dsDNA and the fluorescence decreases on release from the DNA as strand separation occurs. This type of probe may not be suitable for low extents of reaction, as it relies on measuring a small decrease of fluorescence against a large background. In addition, dyes that bind to double stranded DNA may inhibit enzymes acting on such DNA.
Obtaining a quantitative signal change combined with specificity for the ssDNA and discrimination against other similar molecules, such as dsDNA, that may be present in the assay provides a considerable scientific challenge.